Response to interferon in patients infected with HCV has been variable. Recent studies suggested a region, termed IFN sensitivity determining region (ISDR) in the HCV NS5A gene, that are associated with resistance to interferon. The NS5A has also been shown to be a phosphoprotein, probably playing an important role in viral replication and viral-host interaction. Because of the functional importance of NS5A, our laboratory is conducting experiments to characterize its function and identify cellular factors that are the functional targets of this HCV gene product. Using the yeast two hybrid system, several independent clones that interact specifically with NS5A have been identified. One of the NS5A interactors is Bin1, which contains a SH3 domain. The protein-protein interaction between NS5A and Bin1 was confirmed by in vitro binding and in vivo co-immunoprecipitation assays in human hepatoma cells. Deletion and mutation analyses indicated the importance of the SH3 and SH3 binding domains in the interaction between Bin1 and NS5A. Bin1 is a c-Myc-interacting adapter protein with tumor suppressor and cell death properties. Loss of Bin1 may promote malignancy by interfering with the apoptotic pathways. HepG2 cells lack expression of Bin1 and upon infection with adeno-Bin1, these cells undergo apoptosis, as determined by a variety of assays. Expression of the wild-type NS5A but not the mutant NS5A with mutations in the SH3-binding domain inhibits Bin1-induced apoptosis in HepG2 cells. Together, our results suggest that NS5A impairs Bin1-induced apoptosis and exerts its effect on cell growth regulation. Like many viruses encoding gene products interfering with apoptosis, the NS5A and Bin1 interaction may be important for the productive infection of HCV and contribute to the pathogenesis of hepatitis C. Additional experiments are under way to address the functional significance of this interaction. Effort is also being initiated to evaluate the clinical significance of sequence variations in NS5A. Furthermore, the E2 protein has recently been shown to interact with and inhibit PKR (Science 1999; 285:107). The interacting sequences on E2 (PePHD) are variable among the HCV genotypes, possibly underlying the genotypic difference in interferon response. We studied the pretreatment HCV sequence of 34 patients treated with interferon monotherapy. All genotype 1 samples regardless of response had significant homology with the PKR phosphorylation site suggesting resistance to interferon. Three of the six patients with genotype 2 and 3 as well as two of the three with unknown genotype had sequences which varied from the PKR suggesting sensitivity to interferon. Mutations which varied at the 3rd and 9th amino acid position of the PePHD occurred exclusively in sustained responders. Our data suggest that the PePHD plays a role in determining interferon sensitivity although the final response is probably multifactorial. Detailed characterization of this virus-cell interaction and correlation to clinical disease may contribute to our understanding of HCV replication and mechanisms of hepatocellular injury.